Staphylococcus aureus is an important gram-positive human pathogen causing a variety of human diseases in both hospital and community settings. This bacterium is so closely associated with prophages that it is rare to find S. aureus isolates without prophages. Of the staphylococcal phages, the beta-hemolysin (hlb)-converting phage is the most successful phage; it is found in more than 90% of clinical isolates of S. aureus. The phage encodes multiple virulence factors such as exotoxins and immune modulatory molecules, which can inhibit human innate immune responses. Several lines of evidence, however, suggest that staphylococcal prophages increase bacterial virulence by additional mechanisms other than providing virulence factors:1) Transposon insertion in prophage regulatory genes, but not in the genes for virulence factors, reduces S. aureus killing of Caenorhabditis elegans; 2) Although deletion of individual virulence genes encoded by the hlb-converting phage ?NM3 had no effect on the staphylococcal virulence in murine model, deletion of the entire ?NM3 reduced the staphylococcal virulence; 3) The deletion of ?NM3 not only abolished the production of alpha-toxin, an important virulence factor encoded by the chromosome, but also altered the extracellular protein profile. In this research proposal, we test the hypothesis that prophages can increase staphylococcal virulence not only by providing virulence factors but also by altering bacterial gene expression. To test the hypothesis, we will conduct the following specific aims: 1) Determine the global effects of ?NM3 on bacterial gene expression; and 2) Identify genetic determinants involved in the ?NM3-mediated virulence increase and activation of alpha-toxin expression. The results from these studies have the potential to change our current view on the role of phages in bacterial pathogenesis and facilitate our understanding of staphylococcal pathogenesis and the development of therapeutic agents against the important human pathogen. PUBLIC HEALTH RELEVANCE: This proposal will determine how the beta-hemolysin converting phage ?NM3 increases the pathogenic potential of Staphylococcus aureus. The results from these studies will lead us to understand the role of prophages in diseases caused by S. aureus, and how we can make S. aureus more susceptible to immune responses by blocking the phage contribution mechanism.